The recent discovery of two translation initiation factor IF3s, IF3a and IF3b, in the cyanobacterium Fremyella diplosiphon provides an exciting long-term opportunity to better understand how these proteins regulate gene expression at the level of transcriptional attenuation and translation. IF3s are an essential component of the translation initiation process, and therefore cell survival, in many bacteria such as Escherichia coli. However, a genetic screen has revealed that IF3a in F. diplosiphon is not essential for viability, and analysis of its sequenced genome uncovered a second gene encoding an IF3 (IF3b). In addition, both IF3a and IF3b complement an E. coli strain lacking IF3, indicating that both are functional IF3 proteins. Furthermore, IF3a regulates the abundance of transcripts from the cpeC operon at the post-transcriptional level in response to changes in ambient light color. To understand the role of these two IF3s in translational regulation, I propose to initially use ribosome profiling of wild-type strains, strains lacking IF3a, and strains lacking IF3b to identify the mRNAs that are being translated in each strain. In addition, I propose to use deep sequencing of the total mRNA in each strain to study the possible roles of IF3a and IF3b in the genome-wide regulation of transcript abundance in this organism. The combined analyses of global translational activity and transcript levels in these strains will provide a comprehensive view of how IF3a and IF3b affect central cellular processes within F. diplosiphon. These functional studies will be complemented by structural studies. The existence of IF3a and IF3b in this organism provides a unique opportunity to conduct a comparative analysis of their structures using X-ray crystallography and/or NMR to relate any structural differences to differences uncovered in the functional analysis. Overall, this research will provide important insights into the relationship between structure and function for this important class of proteins, results that have not been possible to obtain from any other group of eubacteria, which contain a single, essential IF3. Translation is an important cellular process, and the results of my research will expand our understanding the role of the protein IF3 in bacterial translation initiation. Because IF3 is known to be an essential protein in bacteria and has a structure that is distinct from its equivalent eukaryotic protein, called eIF1, the study of how IF3 function relates to its structure has the potential to lead to the development of novel antibiotics, which is important as more bacteria become resistant to known antibiotics. PUBLIC HEALTH RELEVANCE: Translation is an essential cellular process, and the results of my research will expand our understanding the role of the protein IF3 in bacterial translation initiation. Because IF3 is known to be an essential protein in many bacteria and has a structure that is distinct from its equivalent eukaryotic protein, called eIF1, the study of how IF3 function relates to its structure has the potential to lead to the development of novel antibiotics, which is important as more bacteria become resistant to known antibiotics.